The Potential Role of Interferon-regulatory Factor 7 Among Taiwanese Patients with Systemic Lupus Erythematosus

Patients

All patients with SLE enrolled in our study were identified at the National Cheng Kung University Hospital, a medical center located in southern Taiwan. All patients had the research explained to them in detail and fully informed consent was obtained from each patient before blood was drawn. Fifty-one patients, all of whom had fulfilled at least 4 of the criteria of the American College of Rheumatology classification for SLE, were enrolled for the IRF7 mRNA and serum IFN-α study. The mean age of the patients was 36.65 ± 13.52 years with a female to male ratio of 9:1. Patients with a current or recent infection, chronic viral hepatitis, or malignancy were excluded. Sixty-five age-matched and sex-matched healthy hospital employees were used as the control group. At the time of blood drawing for IFN-α and IRF7 determination, the clinical manifestations and disease activity in each patient were assessed according to the Safety of Estrogens in Lupus Erythematosus: National Assessment — Systemic Lupus Erythematosus Disease Activity Index (SELENA-SLEDAI) instrument, which produces a score ranging from 0 to 105; in addition, flare composite scores were also obtained²⁰. Twenty-one (41.2%) patients had lupus nephritis based on either proteinuria > 0.5 g/day or the presence of urinary cellular casts. The presence of an infection, renal stones, or other causes of these symptoms were excluded among patients diagnosed with lupus nephritis. Anti-dsDNA antibodies were detected by the standard Crithidia luciliae indirect immunofluorescence assay. Twenty-eight patients (54.9%) were found to be positive for anti-dsDNA antibodies. Thirty-four patients (66.6%) had low serum C3. Peripheral blood mononuclear cells (PBMC) and serum were prepared from the heparinized peripheral blood by Ficoll-hypaque separation (Histopaque-1077; Sigma, St. Louis, MO, USA).

For SNP study, a total of 184 subjects were enrolled, including 92 in the SLE group and 92 in the control group. There were only 4 men in the SLE group; they were matched by 4 in the control group. The median age was 38.0 years (Q1, Q3: 30.3, 46.0 yrs) in the control group and 36.0 years (26.0, 45.0 yrs) in the SLE group.

Serum IFN-α level

We measured the IFN-α level in the serum samples with the Human IFN Alpha Serum Sample ELISA Kit (PBL Medical Laboratories, Piscataway, NJ, USA) following the manufacturer’s protocol. The detection range of IFN-α by the kit was 0.1–100 pg/ml. Samples and controls (human IFN-α standard from the kit) were serially diluted in an anti-IFN-α antibody-coated microtiter plate, reacted with anti-IFN-α antibody, reacted with horseradish peroxidase-conjugated secondary antibody, developed with tetramethyl-benzidine, and read on a microtiter plate reader at 450 nm. To calculate the concentration of IFN-α, the serum sample data were compared to a standard curve generated from the controls.

IRF7 mRNA expression by real-time polymerase chain reaction (RT-PCR)

Aliquots of the blood samples were treated with Blood RNA Stabilization Reagent (Qiagen, Chatsworth, CA, USA) to stabilize the total RNA, and then centrifuged to pellet nucleic acids. The pellet was washed, resuspended, incubated in buffered proteinase K (Qiagen, Cat. No. 19133) to digest any proteins present, and then centrifuged through a Shredder spin column (Qiagen, Cat. No. 79654) to homogenize the cell lysate while removing residual cell debris. The supernatant was mixed with chloroform in a new microfuge tube and centrifuged. The RNA sample was then mixed with ethanol and centrifuged through an RNeasy Mini spin column (Qiagen, Cat. No. 74106), which was then washed multiple times, treated with DNase I (Qiagen, Cat. No. 79254) to digest any DNA present, and finally eluted into nuclease-free water.

Reverse transcription

To prepare a complementary DNA (cDNA) pool from each RNA sample, total RNA (5 μg) was reverse-transcribed using Moloney Murine Leukemia Virus reverse transcriptase (Promega, Madison, WI, USA) and the resulting samples were diluted 40 times with DNase-free water. Each cDNA pool was stored at −20°C until RT-PCR analysis. Specific oligonucleotide primer pairs were selected from the Roche Universal Probe Library for the RT-PCR assays. The specificity of each primer pair was validated by performing an RT-PCR reaction using a common reference RNA (Stratagene, La Jolla, CA, USA) as a template, and the size of the PCR product was checked using a DNA 1000 chip (Agilent, Santa Clara, CA, USA) run on a Bioanalyzer 2100 (Agilent). Primer pairs generating the predicted product size and no other side-product were chosen to conduct the following RT-PCR reaction. RT-PCR reactions were performed on the Roche LightCycler Instrument 1.5 using the LightCycler FastStart DNA MasterPLUS SYBR Green I kit (Roche Cat. 03 515 885 001, Castle Hill, Australia). Briefly, 10 μl reactions were used that contained 2 μl Master Mix, 2 μl of 0.75 μM forward primer and reversed primer, and 6 μl cDNA sample. Each sample was run in triplicate. The RT-PCR program consisted of 95°C for 10 min followed by 40 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 10 s. At the end of the program a melt curve analysis was done. At the end of each RT-PCR run, the data were automatically analyzed by the system and an amplification plot was generated for each cDNA sample. The LightCycler 3 Data analysis software automatically calculated the Cp value for each reaction. The fold expression or repression of the target gene relative to the internal control gene, peptidylprolyl isomerase H in each sample, was then calculated using the formula: 2−ΔΔCp where ΔCp=Cp target gene−Cp internal controland ΔΔCp=ΔCp test sample−ΔCp control sample

IRF7 polymorphism genotyping

We investigated 2 IRF7 polymorphism sites, NCBI Reference Sequences NM_004031.2 [IRF7 mRNA 776 G>G/A(R); rs1061501] and NCBI Reference Sequences NM_004031.2 [IRF7 mRNA 984 A.A./G(R); rs1061502]. The rs1061501 polymorphism is a synonymous change, while the rs1061502 polymorphism results in a missense mutation. ABI Taqman assays (VIC-C allele; FAM-T allele) were performed for the SNP study, the reverse sequence for the rs1061501 polymorphism is GGT CGG CCG GGT CCC CCG AGT TATC [C/T] CGC AGCA TCA CGA AGC GAC GGG TGC, and ACT GCC TGG AGC AGG AGG TCC CCCT [C/T] GT CAC CAG CTG GGG CAG GGA GGG GG for the rs1061502 polymorphism.

ABI TaqMan SNP genotyping assay

TaqMan SNP Genotyping Assays (Applied Biosystems Inc., Foster City, CA, USA) were used to determine genotypes using archived genomic DNA isolated from PBMC. Assays-on-Demand primers and probes were obtained from Applied Biosystems. The TaqMan assays were performed following the manufacturer’s instructions on an MX3000P instrument (Stratagene). The PCR was carried out in a total reaction volume of 25 μl, which contained 12.5 μl of 2× TaqMan Universal PCR Master Mix, 1.25 μl of 20× TaqMan SNP Genotyping Assay Mix, 1 μl containing 1–20 ng DNA sample with ddH₂O added to give a 25 μl final volume. The PCR program was as follows: initial denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 92°C for 15 s, and annealing/extension 60°C for 1 min. At the end of each PCR run, the products were automatically analyzed by the system and an amplification plot was generated for each DNA sample. Each genotyping plate contained positive and negative controls. About 10% of the samples were duplicated to assure the quality control of the genotyping.

Statistical analysis

Normally distributed continuous data were compared by independent 2-sample t-tests and categorical variables were compared by chi-squared tests. When the data were far from normality, Mann-Whitney U tests were applied. Continuous data were displayed as mean ± SD. Categorical data were represented by number (%), and nonparametric data were presented as medians (interquartile range). Statistical assessments were 2-sided and evaluated at the 0.05 level of significant difference. Statistical analyses were performed using SPSS 15.0 statistical software (SPSS, Chicago, IL, USA).